The program described in this application is aimed at determining the causative factors involved in the blistering skin disease pemphigus. These studies are based on our initial findings that pemphigus patients contain circulating autoantibodies directed against components associated with the desmosomes of stratified epithelial tissues. The specific aims of this proposal are to continue these studies by determining the precise cellular sites recognized by these autoantibodies using morphological, biochemical, immunochemical and physiological methods. The morphological methods which we will use will include immunofluorescence, immunogold localization for ultrastructural studies, and double label immunofluorescence techniques. These will be applied to both thin cryostat sections of various tissues including human biopsy material and cultured keratinocytes. The biochemical analyses will involve the isolation of desmosomes. These desmosomes will be used as substrates for the 'Western' immunoblotting procedure which will permit the identification of pemphigus autoantibodies which recognize desmosome associated components. Affinity procedures will be used to purify these antibodies from human serum samples and the resulting monospecific antibodies will, in turn be used to determine if these autoantibodies are factors involved in the loss of cell cohesion in the disease. Other studies will be aimed at determining whether or not there are different autoantibodies involved in the major variants of the disease, pemphigus vulgaris and pemphigus foliaceus. Attempts will be made to prepare rabbit antibodies against desmosome associated proteins recognized by autoantibodies in the two variants of the disease and determine whether or not these can mimic pemphigus autoantibodies. Finally we will attempt to prepare monoclonal human antibodies by fusing peripheral blood lymphocytes from blood samples of patients with pemphigus to a human-mouse heteromyeloma line. The long term goal of these studies is to determine the causative factors in pemphigus and to provide new insights into the cellular/molecular basis of the disease. Ultimately this information may be important in determining the therapeutic regimens of pemphigus patients.